Background: The increasing incidence of oral squamous cell carcinoma (OSCC) combined with its poor and unchanged prognosis motivates a need for research into the aspects of tumor biology such as the expression of adhesion molecules. Alterations in the properties of adhesion molecules could play a pivotal role in the development and progression of invasive cancer and distant metastasis. Aim: This study aims to quantify the expression of cluster of differentiation 44 (CD44) in the different grades of OSCC. Materials and Methods: Immunohistochemical staining for CD44 was performed in 10 tissue sections each of normal mucosa, well-differentiated OSCC, moderately differentiated OSCC, and poorly differentiated OSCC. CD44 positivity was analyzed quantitatively in 10 representative fields of each section under 20× magnification. Statistical Analysis Used: Statistical analysis was performed using Statistical Package for Social Sciences software, version 20.0 (SPSS Inc., Chicago, IL, United States). Results: The mean value of the number of positive cells in controls was 745 ± 68.17, in well-differentiated OSCC was 694.2 ± 145.47, in moderately differentiated OSCC was 349 ± 78.40, and in poorly differentiated OSCC was 108.8 ± 34.39. Conclusion: The results of this study suggest an altered expression of CD44 in OSCC with weak immunostaining in poorly differentiated squamous cell carcinoma. Thus, it can be inferred that the loss of cell adhesion, correlated to the decrease of CD44 expression, might be of value in determining the progression of OSCC.

Head and neck cancer is a major burden in Asia particularly in India, where more than 200,000 new cases are diagnosed each year. Moreover, 7.8% of the global cancer burden and 8.33% of global cancer deaths are contributed by the Indian subcontinent.[1] Oral cancer accounts for 30% of all the cancers in the country.[2] Squamous cell carcinoma accounts for 95% of the malignant tumors of the oral cavity. More than 60% of the patients present with an advanced stage of the disease.[3] It is the sixth most common cancer in the world, eighth most common cancer in men, and fifth most common cancer in women.[4] As per the global cancer statistics published in 2012, the incidence of oral squamous cell carcinoma (OSCC) is estimated at 300,400 new cases and 145,400 deaths annually.[5] The overall 5-year survival rate is <50% and has not improved significantly over the last three decades.[6] Despite the accessibility of oral cavity for visual examination along with well-defined clinical diagnostic features for oral cancers, they are being detected in their advanced stages. In the Indian scenario, 60–80% of patients present with an advanced stage of disease as compared to 40% in developed countries.[7]

Carcinogenesis is a multistep process, which alters the genetic events within signal transduction pathways that govern normal cellular physiology such as cell division, differentiation, senescence, and adhesion. As the alterations or mutations in these pathways get accumulated, the cells become functionally independent from the surrounding normal epithelial cells with enhanced ability to proliferate and invade or metastasize to distant sites. The accumulation of such changes will correlate to the histologic grade of the differentiation of cancer and also reflects on the prognosis.[8]

The increasing incidence of OSCC, combined with its poor and unchanged prognosis, motivates a need for research into the aspects of tumor biology such as the expression of adhesion molecules. Alterations in the properties of adhesion molecules could play a pivotal role in the development and progression of invasive cancer and distant metastasis. The loss of cell–cell adhesion allows malignant cells to escape from their site of origin, degrade the extracellular matrix, and acquire a motile and invasion phenotype that can metastasize.[9]

The cluster of differentiation 44 (CD44) is a transmembrane cell adhesion molecule involved in cell-to-cell and cell-to-matrix interactions by binding with hyaluronan, extracellular matrix proteins, and growth factors.[10] Dalchau et al. in 1980 first described CD44 as brain-granulocyte-T lymphocyte antigen.[11] It was first identified on the lymphocytes having functions essential for cell homing and is encoded by a gene located on the short arm of chromosome 11.[12] CD44 consists of a cytoplasmic domain and a transmembrane domain. It is constituted by 20 exons, in which the first five and the last five exons are constant, and the remaining 10 exons result in the generation of a variable region.[13] CD44 has various isoforms that exist due to the splicing of variable exons, which encode the proximal portions of the extracellular cytoplasmic domain.[14]

The invasion and dissemination of OSCC requires active cell migration through the extracellular matrix with a remodeling of intercellular adhesions.[15] CD44 is thought to undergo structural and functional alterations during malignant transformation, which lends the cancer cells to detach from the site of tumor growth and invade the surrounding tissues. Hence, CD44 deserves considerable attention with respect to its adhesive, locomotive, and growth-transducing functions among the cancer cells.[16] CD44 is also a cancer stem cell marker with two distinct phenotypes such as: (i) The epithelial–mesenchymal transition phenotype cancer stem cells that express proteolysis-resistant CD44s isoform and (ii) The cancer stem cells with epithelial phenotype that expresses trypsin-sensitive CD44v isoforms. This results in the loss of CD44 expression in epithelial phenotype cancer stem cells and a higher expression in the epithelial–mesenchymal transition phenotype during isolation.[17]

The field of human cancer research is rapidly advancing, and the application of molecular biology tools such as immunohistochemistry made the diagnosis much easier. This study aimed to assess the immunohistochemical expression of CD44 in the different grades of OSCC and to evaluate its role as an indicator of cancer progression.

Materials and Methods

This study was performed using 10 formalin-fixed, paraffin-embedded tissue blocks of each category, which were diagnosed histologically as well-differentiated, moderately differentiated, and poorly differentiated OSCC, and were retrieved from the archives of the Department of Oral Pathology and Microbiology, SIBAR Institute of Dental sciences, Guntur. As controls, 10 formalin-fixed, paraffin-embedded tissue blocks of the normal oral mucosa over the impacted third molars was obtained from the patients undergoing surgery for impactions after informed consent. Ethical approval for this study was obtained from the Ethical Committee of SIBAR Institute of Dental Sciences, Guntur, on 24th December 2013 (Ref.no.20/IEC-SIBAR/2013). The grading of OSCC was performed based on the criteria proposed by the World Health Organization in 2005.

Samples were categorized into four groups as follows:

Group I—Controls (10).

Group II—Well-differentiated OSCC (10).

Group III—Moderately differentiated OSCC (10).

Group IV—Poorly differentiated OSCC (10).

Control group comprised apparently normal healthy oral mucosa, which was obtained from patients without any tobacco smoking or chewing habits, alcohol consumption, and any clinically obvious lesions.

Serial sections having a thickness of 4 μm were prepared. The sections of Groups I–IV were first subjected to routine hematoxylin and eosin examination to reconfirm the diagnosis. Later, other sections of all the four groups were subjected to immunohistochemical analysis using anti-CD44 antibody. For immunohistochemical staining, sections were carefully fixed on microslides coated with ploy-l-lysine. The tissue was deparaffinized by giving two dips of 10 min each in fresh xylene. The rehydration of the tissue was performed by giving three dips for 5 min each in 90, 80, and 70% alcohol. Thereafter, the tissue was placed in a distilled water bath and was not allowed to dry. Tissue sections were dipped in buffer solution prepared by mixing 1.21 g of Tris Ethylene diamine tetra acetic acid (EDTA) buffer with 0.37 g of EDTA and incubated at 850 W for 5 min, 600 W for 10 min, and 400 W for 5 min in a microwave oven. The slides were allowed to cool and then placed in a wash buffer prepared by adding 950 ml of distilled water with 50 ml of buffer. Excess buffer from the slides was removed using tissue paper. Then the tissue specimen was covered with 50 μl of hydrogen peroxide, incubated for 5 min, and gently washed twice with phosphate buffered saline. The tissue specimen was then covered with primary antibody—a monoclonal anti-CD44 antibody—incubated for 1 h at room temperature and gently washed twice with phosphate buffered saline. The secondary link antibody was then added. Specimens were incubated for 30 min at room temperature and given three gentle rinses with phosphate buffered saline. The substrate chromogen solution prepared by mixing 1 ml of substrate buffer and a drop of diaminobenzidine was incubated for 5 min at room temperature and later gently rinsed with phosphate buffered saline. Then the slides were counterstained in a bath of hematoxylin for 2–5 min and washed under tap water for 5 min.

Positive CD44 expression was seen as a light brown stain in the cell membrane. All stained areas demonstrating positivity for CD44 were identified at a magnification of 20×, and the number of positively stained cells was counted on 10 representative areas of the section, in a minimum of 100 cells per field [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8]. The CD44 positivity was estimated according to the criteria of Simionescu et al. as follows:[18]

The collected data were entered in theExcel spreadsheet, and statistical analysis was performed using the Statistical Package for Social Sciences software, version 20.0 (SPSS Inc., Chicago, IL, United States). The normal distribution of the number of positive cells was determined by “Kolmogorov–Smirnov test.” A comparison of the four groups with respect to the percentage of positive cells, as well as the scores, was performed using “Kruskal–Wallis analysis of variance (ANOVA) test.” A pairwise comparisonof the three grades of OSCC and the controls was performed using “Mann–Whitney U test.”

Results

A total of 30 cases of OSCC, divided into three groups with 10 samples each of well-differentiated, moderately differentiated, and poorly differentiated squamous cell carcinomas and 10 samples of controls were taken for the study. All the groups were subjected to immunohistochemical analysis using anti-CD44 antibody. The number of immunopositive cells was evaluated in each case, and a score was given accordingly.

The mean value with respect to the percentage of positive cells was 74.5 ± 6.82 in Group I (controls), 69.42 ± 14.55 in Group II (well-differentiated OSCC), 34.9 ± 7.84 in Group III (moderately differentiated OSCC), and 10.88 ± 3.44 in Group IV (poorly differentiated OSCC). Statistical analysis was performed using “Kruskal–Wallis ANOVA test,” and a high statistically significant difference of mean was obtained (P = 0.00001) [Table 1].

Table 1: Comparison of four groups with respect to the percentage of positive cells by Kruskal–Wallis ANOVA test

A pairwise comparison of the four groups with respect to the percentage of positive cells was performed using “Mann–Whitney U test.” A high statistical significance was noticed on comparing Group I and Group III, Group I and Group IV, Group II and Group III, Group II and Group IV, and Group III and Group IV (P = 0.0002). However, a comparison of Group I and Group II did not show any statistical significance (P = 0.3258) [Table 2].

Table 2: Pairwise comparison of four groups with respect to the percentage of positive cells by Mann–Whitney U test

A comparison of the four groups with respect to the score given based on the number of immunopositive cells was performed using “Kruskal–Wallis ANOVA test.” Of the 10 cases in Group I (controls), five (50%) had Score 3, and the other five (50%) had Score 4. One case (10%) of Group II (well-differentiated OSCC) had Score 2, five cases (50%) had Score 3, and four cases (40%) had Score 4. In Group III (moderately differentiated OSCC), nine cases (90%) had Score 2, and one case (10%) had Score 3. Five cases (50%) of Group IV had Score 0, and the other five cases (50%) had Score 1. Of the total 40 samples, Score 0 and Score 1 were seen in five cases each, with all being poorly differentiated squamous cell carcinoma. Score 2 was seen in 10 cases, with nine being moderately differentiated OSCC and one being well-differentiated OSCC. Score 3 was seen in 11 cases, with five each in controls and well-differentiated OSCC and one in moderately differentiated OSCC. Score 4 was observed in nine cases, with five and four each in controls and well-differentiated OSCC, respectively. A high statistical significance (P = 0.00001) was obtained [Table 3].

A pairwise comparison of the four groups with respect to score was performed using “Mann–Whitney U test.” A high statistical significance was noticed on comparing Group I and Group III (P = 0.0003), Group I and Group IV (P = 0.0002), Group II and Group III (P = 0.0015), Group II and Group IV (P = 0.0002), and Group III and Group IV (P = 0.0002). However, there was no statistical significance between Group I and Group II (P = 0.5708) [Table 4].

Table 4: Pairwise comparisons of four groups with respect to score by Mann–Whitney U test

Oral cancer being the sixth most common cancer and with OSCC being the most common among the general population, they represent a major health problem in many parts of the globe.[19] Carcinogenesis is a multistep process resulting from the accumulation of genetic alterations that occur even before the development of a malignant phenotype. As the tumor progresses, cells undergo further mutations, leading to an increased heterogeneity of the tumor cell population. The cells detach from the tumor mass due to the loss of adherence property and invade the neighboring tissues. The detached cells enter the blood and lymph, through which they are transported to distant sites (metastasis) and lead to the development of secondary tumors.[20] In this study, the immunoexpression of CD44 was assessed among the various grades of OSCC.

In this study, a decline in the expression of CD44 was observed with the increasing grades of OSCC when compared with the normal oral mucosa, which was in accordance with studies conducted by Mende et al., Kuo et al., Satoa et al., Carinci et al., Gonzalez-Moles et al., Mostaana et al., and Krump and Ehrmann.[21],[22],[23],[24],[25],[26],[27] A strong expression of CD44 in the normal epithelium when compared with the OSCC indicates that CD44 is required for the maintenance of epithelial structure as a whole by the homing of the epithelial cells. The loss of CD44 expression favors the invasiveness of tumor cells by emancipating the cells from their attachment with the neighboring cells. It plays a crucial role in the behavior of malignant tumors.[28]

The mean value of CD44 immunopositive cells in this study is higher in well-differentiated squamous cell carcinoma when compared to moderately and poorly differentiated squamous cell carcinomas. Poorly differentiated squamous cell carcinoma cases showed diminished CD44 expression. A correlation is made with the immunostaining degree, as given by the total number of positive cells in each case. The immunostaining degree is proportional to the grade of the differentiation of OSCC. All cases of well-differentiated squamous cell carcinoma expressed the highest immunostaining score of 3 and 4 with 50–75% and over 75% positive cells, respectively. Moderately differentiated squamous cell carcinoma cases presented a lower immunostaining score of 2 with 25–50% of immunopositive cells. When poorly differentiated squamous cell carcinoma was evaluated, half the cases presented a weak immunostaining score of 1 with 10–25% positive cells, and the other half of the cases showed zero degree with <10% of CD44-positive cells. We can assess that a total absence of score or Score 1 on immunostaining was associated with undifferentiated OSCC having an unpredictable prognosis. These findings are in accordance with the studies of Salmi et al., Bahar et al., Kunishi et al., Soukka et al., Ue et al., Stoll et al., Kanke et al., Fonseca et al., Simionescu et al., Hedesiu et al., da Cruz et al., and Hema et al.[29],[30],[31],[32],[33],[34],[35],[36],[37],[38],[39],[40]

The restricted expression of CD44 in various grades of OSCC was related to the degree of cellular proliferation and differentiation. The decrease in the immunostaining expression of CD44 with increase in the grade of tumor may suggest a loss of cell–cell adhesion, thereby contributing to the easy detachment of cells from a rigid configuration. The varying pattern of expression in the different grades of OSCC was related to the presence of pleomorphic cells. Highly pleomorphic cells with a weak resemblance to the parent tissue were present in poorly differentiated squamous cell carcinoma, and hence, a weak CD44 immunostaining is associated with poorly differentiated squamous cell carcinoma. CD44 also signals for the orientation of epithelial cells that differentiate and migrate upward. These events are switched off as the cells reach their end differentiation and are disclosed from their intercellular junctions. CD44 downregulation might reflect an early cellular change from normal cell–cell and cell–matrix interactions to a bizarre heterotypic cell surface adhesion property that predisposes the cells to attain invasiveness.[41] The normal oral epithelium showed an extensive expression of CD44 in the basal and parabasal cells with negative expression in the corneal and subcorneal layers. In OSCC, CD44 is localized to the basal and parabasal layers and around the peripheral cells of the tumor nests. Similar findings were observed in the study by Andratschke et al.[42] The strong expression of CD44 observed in the generative cells of the epithelium implicate that these cells could be the targets for malignant transformation.[43]

In this study, the expression of CD44 in the normal oral epithelium was detected as membranous staining localized on to the surface of the epithelial cells. On evaluation of the OSCC cases, apart from the membranous staining, cytoplasmic labeling was also evident. This could be due to the interaction of the cytoplasmic domain of CD44 with cytoskeletal linker proteins such as ezrin and ankyrin through which it can mediate cell migration on hyaluronan.[44],[45]

The disturbances in the expression of CD44 adhesion molecule during the process of oral carcinogenesis can be associated with unfavorable prognosis and an increased probability of developing metastasis. Therefore, poorly differentiated carcinomas with a weak immunostaining of CD44 can present with unpredictable prognosis.

Conclusion

The results of the present study suggest an altered expression of CD44 in OSCC with weak immunostaining in poorly differentiated squamous cell carcinoma. It implies that the adhesive functions of CD44 in cell–cell and cell–matrix interactions are needed in the maintenance of the normal architecture of the epithelium. The downregulation of CD44 could pave way for the cells to detach and invade. The expression of CD44 could also elucidate the difference in the biological behavior of the different grades of OSCC. Thus, it can be inferred that the loss of cell adhesion correlated to the decrease of CD44 expression might be of prognostic value in the evolution of OSCC.